A remarkable new deep-sea nereidid (Annelida: Nereididae) with gills

Nereidid polychaetes are well known from shallow marine habitats, but their diversity in the deep sea is poorly known. Here we describe an unusual new nereidid species found at methane seeps off the Pacific coast of Costa Rica. Specimens of Pectinereis strickrotti gen. nov., sp. nov. had been observed dating back to 2009 swimming just above the seafloor at ~1,000 m depth but were not successfully captured until 2018. Male epitokes were collected as well as a fragment of an infaunal female found in a pushcore sample. The specimens were all confirmed as the same species based on mitochondrial COI. Phylogenetic analyses, including one based on available whole mitochondrial genomes for nereidids, revealed no close relative, allowing for the placement of the new species in its own genus within the subfamily Nereidinae. This was supported by the unusual non-reproductive and epitokous morphology, including parapodial cirrostyles as pectinate gills, hooked aciculae, elfin-shoe-shaped ventral cirrophores, and elongate, fusiform dorsal ligules emerging sub-medially to enlarged cirrophores. Additionally, the gill-bearing subfamily Dendronereidinae, generally regarded as a junior synonym of Gymnonereidinae, is reviewed and it is here reinstated and as a monogeneric taxon.

The deep sea encompasses a diverse, vast mosaic of understudied and poorly sampled habitats [16].Several surveys that have sampled deep waters from distinct regions suggest polychaete biodiversity is severely underestimated [17][18][19], rendering our knowledge of it limited [20][21][22].We can assume, therefore, that oceanic depths still host a vast number of yet undescribed nereidid species, although it is noteworthy that there has been a relatively continual discovery of new deep-sea nereidids since about the 1960s (e.g., [6, 10-12, 14, 15, 23-36]. While deep-water nereidids continue to be discovered, information about their behavior and inference about morphological adaptations has been scarce.According to Fauchald [11,12], deep-sea nereidids share a few unusual prostomial and parapodial features when compared to shallow-living relatives, such as the absence of eyes, prolonged appendages and chaetae, and posterior chaetigers with extended notopodia and elongated neuropodia.Notably, the reduction of eyes and the elongation of parapodial appendages and chaetae fall under the general specialization to aphotic environments of some subterranean and cave (troglobiotic) nereidids placed in Namanereidinae Hartman, 1959 [37-39].These morphological adaptations, inherent to the 'darkness syndrome' [40], have been shown for a few other polychaete clades containing cave and deep-sea members (e.g., Scalibregmatidae Malmgren, 1867 [41] and Aphroditiformia Levinsen, 1883 [42][43][44][45]), suggesting that these are also convergent evolutionary traits for nereidids living at aphotic depths.
Genetic tools have been instrumental in developing a better understanding of the diversity among nereidids.Several new species or previously synonymized ones have been described, reinstated, or delimited through the integration of morphological data and molecular markers of apparently cryptic species (e.g., [46][47][48][49][50][51][52][53][54][55][56][57]).Molecular data has also been utilized to investigate phylogenetic relationships at a broader scale among nereidid taxa [15,57,58].Recently mitochondrial genomes (mitogenomes) have been sequenced for a variety of nereidid species (e.g., [59][60][61][62][63][64][65][66], providing a rich dataset to assess phylogenetic relationships with more confidence.Alves et al. [67] assessed the monophyly and phylogenetic relationships of the presently accepted subfamilies and provided an ancestral state reconstruction of pharyngeal structures using mitogenomic data.They rejected the monophyly of the subfamilies Nereidinae de Blainville, 1818 and Gymnonereidinae Banse, 1977 as currently recognized and revealed that the occurrence of papillae and paragnaths may not be reliable features to diagnose major groups.We revisit the nereidid subfamily issue here with regards to Dendronereidinae Pillai, 1961.This study describes a new, morphologically unusual, nereidid species belonging to a new genus using specimens found near methane seeps at ~1,000 m depth off Costa Rica in the eastern Pacific.This new species is remarkable for its dorsal and ventral anterior parapodial cirri, modified as gills, and hooked-shaped posterior aciculae, both unique features among Nereididae.Its mitochondrial genome was sequenced as well as those of two shallow-water nereidid species.This allowed for a new mitogenomic analysis to evaluate the position and relationships of the new genus within Nereididae.

Mitochondrial genome assembly and annotation
Sequence reads were trimmed and cleaned with Trimmomatic v. 0.39 [70] before assembly with MitoFinder v. 1.4 [71] with The Invertebrate Mitochondrial Code (NCBI; transl_table = 5) used to translate the 13 protein-coding genes (PCGs).Complete records for all RefSeq annelid mitogenomes available on NCBI GenBank were used as the MitoFinder reference file, with MEGAHIT v. 1.2.9 [72] metagenomic assembler and Arwen v.1.2.3 [73] tRNA gene annotator selected for the assembly parameters.Resulting mitochondrial genes recovered in MitoFinder [71] contigs were checked for contamination using NCBI's Nucleotide BLAST.The MITOS Web Server [74] was used for mitogenome annotation.Geneious Prime v. 2022.2.2 [75] was used to manually finalize annotations, extract the 13 PCGs and two ribosomal RNA genes (rRNAs), and translate the PCGs into amino acids.Nuclear 18S rRNA gene sequences (18S) were also mined out for the three species: using BBMap v. 38.87 [76], post-Trimmomatic paired-end reads were interleaved with the included reformat.shscript.Publicly available and closely related nereidid 18S sequences were extracted in FASTA format from NCBI.Interleaved FASTQ files for each of the three species were mapped individually to the nereidid 18S sequences using Minimap2 v. 2.22 [77,78], then SAMtools v. 1.13 [79] was used to extract the resulting mapped reads.The Map to Reference tool in Geneious Prime v. 2022.2.2 [75] was used to individually map the FASTQ mapped reads to the nereidid 18S FASTA file for read coverage visualization and consensus sequence extraction.Newly assembled and annotated mitogenomes obtained in this study were deposited in GenBank with accession numbers OL782598-600 and the 18S sequences as OR437939-41 (Table 1 and S1 Table).

Haplotype network and phylogenetic analyses
A haplotype network using COI data from the four specimens of the new species was generated with PopART v. 1.7 [80] using the TCS algorithm [81,82].A maximum likelihood (ML) phylogenetic analysis of concatenated COI, 16S rRNA, and 18S rRNA DNA sequences from across Nereididae (S1 Table ) was analyzed, with each gene under the model GTR+FO+I+G4, chosen with ModelTest-NG v. 0.1.7[83] and executed using raxmlGUI v. 2.0.10 [84].The best ML tree was chosen after 100 ML runs (seed 581027) and support was assessed via thorough bootstrapping (1,000 pseudoreplicates).

Morphology
Methods for measurements of specimens (total body length, TL; length to chaetiger 15, L15; body width to chaetiger 15, W15), counting of structures and ridge patterns on the proboscis, and body dissections were explained elsewhere [87].For practical purposes, decimal numbers are used when measurements between two structures exceed one unit (e.g., 1.3 times, 2.5 times, twice).In contrast, written fractions were used when those measurements were less than one unit (e.g., half, two-thirds, four-fifths).
Light microscopy observations were made using both stereo and compound microscopes.Specimens were photographed alive using a Canon EOS M5 camera.Preserved specimens were photographed with a Nikon D5100 or Canon Rebel T7 camera mounted on both the compound microscope and stereomicroscope.Some images were generated using stacks using Helicon Focus1 6 (Method C) or manually through Adobe Photoshop1 CS6 (for chaetae).Parapodia of one specimen were processed for scanning electron microscopy (SEM) with a Zeiss Evo10 scanning electron microscope.Figure backgrounds were cleaned and darkened or lightened as necessary without manipulating the actual specimen.Parapodia were shown in anterior views unless otherwise stated.
Descriptions of the species are based on the holotype morphology unless otherwise stated.The methods performed, terminology, and standardized definitions established for overall nereidid features either newly proposed, partially readapted, or based upon references cited in Villalobos-Guerrero et al. [4] were followed.These authors proposed a division of the dorsal ligule into two main regions: proximal and distal.However, we consider that the proximal dorsal ligule ([4]: Fig 1C , dld) is instead the basal part of dorsal cirrus, namely the dorsal Table 1.Mitochondrial sequences used for the phylogenetic analysis.Three new mitogenomes were generated for this study (bold).Also, four COI sequences generated for the holotype and three paratypes of Pectinereis strickrotti gen.nov., sp.nov.are listed.Note that the name Laeonereis cf.pandoensis is used here instead of Laeonereis culveri (Webster, 1879) since the specimen was collected in Brazil.The correct spelling for sequences deposited in GenBank as Tylorrhynchus heterochaetus is actually Tylorrhynchus heterochetus., and among others.All these taxa share the distal placement of the dorsal cirrostyle, which is easily recognized by the articulation and thickened basal tegument (sometimes barely evident in Namalycastis), and the reduced dorsal ligule when present on the fully expanded dorsal cirrophores.Nicoll [88] and Kaufmant [94] also found a similar irrigation of the notopodium in A. virens Sars, 1834 and Hediste diversicolor (Mu ¨ller, 1776) (both as Nereis), respectively.However, the position of notopodial vessels and the innervations of capillaries were different; the dorsal cirrophore was only slightly enlarged, and the dorsal cirrostyle was sited sub-medially on the notopodia.Finally, the relative extension of parapodial structures was described following Villalobos-Guerrero & Carrera-Parra [49].However, the dorsal ligule and the parapodial cirrostyles were measured in comparison with the entire length of the parapodial cirrophore in natatory chaetigers of epitokous specimens.The first and last natatory chaetigers of epitokes were determined by the appearance/disappearance of additional parapodial lobes, particularly the expanded neuropodial postchaetal lobe.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article.This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN.The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information is viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/".The LSID for this publication is: urn:lsid:zoobank.org:pub:9E9C5C-D6-EFE8-4B90-A63F-96A4544AC60B.

Haplotype network and phylogenetic analysis
The four COI sequences obtained from the new species were all unique, but varied by a maximum of less than 1%, five base pairs out of an alignment of 676 bases ( The mitochondrial genome order for all the three newly sequenced taxa was the same as one of the two observed gene orders for Nereididae, identified as Group I by Alves et al. [67].The newly generated mitogenomes of three nereidids, plus those from 23 other species (plus outgroups), resulted in a concatenated sequence alignment of 2,325 sites for the two rRNA genes and 3,829 amino acids for the 13 PCGs.The ML tree (log likelihood = -127521.3745)showed a high bootstrap support (>90%) for many clades (Fig 2B ), though some key nodes were recovered with lower support.Allowing for the additional terminals used here, the results were largely congruent with those of Alves et al. [67].Two major clades were found: Clade I was a well-supported Nereidinae, while Clade II, with relatively low support, consisted of species belonging to Dendronereidinae, Tylorrhynchus, Namanereidinae, and Paraleonnates (Fig 2B).Dendronereidinae was represented by two terminals, Dendronereis chipolini Hsueh, 2019 and Neanthes glandicincta (Southern, 1921), although based on the shallow genetic distance between the individuals, the latter terminal is apparently a misidentification.The placement of Pectinereis strickrotti gen.nov., sp.nov.within Nereidinae was as the poorly supported sister group to a clade comprised of Alitta, Hediste, Nectoneanthes, Perinereis, Platynereis, and Pseudonereis terminals.The newly generated mitogenome for the type species of Nereis, N. pelagica, formed a well-supported clade with Nereis zonata Malmgren, 1867 while Nectoneanthes oxypoda was a well-supported sister group to Alitta succinea (Fig 2B ).

Non-reproductive morphology
Gills.Oxygen exchange in nereidids is taken over by the branchiae, which are present as vascularized parapodial structures [1].Elaborate branchiae, namely gills, develop only in a few genera; they are associated with the dorsal cirrophore and start some distance from the prostomium.The most distinctive and complex gills have been reported in two genera: Dendronereides Southern, 1921, and Dendronereis Peters, 1854.The gills in Dendronereides are arborescent with branched bunches of filaments inserted basally on the cirrophore and above the median ligule.In contrast, those in Dendronereis Peters, 1854 are markedly modified dorsal cirrophores with bipinnate shapes, consisting of branches arising laterally from the primary axis.Enlarged dorsal cirrophores have also been reported as gills in Gymnonereis [95], Namalycastis [89,96], Nereis [97], and Tambalagamia Pillai, 1961 [98], and those highly vascularized should also be treated in the same manner in members of Alitta (see [49]: Figs 2F and 3E; [99]:  5D), as well as some Neanthes with enlarged dorsal cirrophores (see [101]: table II, as shape of dorsal ligule), among others.
The gills in Pectinereis gen.nov.are an exceptional case among nereidids.These respiratory organs are modified dorsal and ventral cirrostyles of anterior chaetigers, with the former thicker and encompassing a slightly higher range of chaetigers.They are pectinate in shape by the 4-11 filaments that arise from the upper edge and vascularized with a main broad blood vessel that runs along the stem and branches out to the filaments.In contrast to nereidids with gills associated to dorsal cirrophores and starting at some distance from the prostomium, these structures in Pectinereis gen.nov.are associated not only with the dorsal but also to the ventral cirrostyles, although with a slightly more restricted distribution in anterior parapodia.
Pectinereis gen.nov.gills may remind the shape of scalloped or swollen cirrostyles of some epitokous nereidids, which are associated with the chemical reception of pheromones [102,103].Nevertheless, the former are short, knob-like structures exclusive of natatory parapodia present typically in males, whereas those in Pectinereis gen.nov.are elongate and digitiform, restricted to the pre-natatory parapodia.Although the swollen cirrostyles of epitokous nereidids and the pectinate cirrostyles of Pectinereis gen.nov.are present in both dorsal and ventral cirri of pre-natatory parapodia, both forms are very different by themselves.Additionally, the swollen cirrostyles are present in up to the first 7-8 parapodia, whereas the pectinate cirrostyles of Pectinereis gen.nov.are present at least in the first 14 parapodia.These structures are not homologs due to their morphology and function.Therefore, the presence of gills can be considered a non-reproductive modification in Pectinereis gen.nov.Dorsal ligule + dorsal cirrophore.When present in nereidids, the dorsal ligule is attached frontally to the dorsal cirrophore, although it seems to be attached to the notoacicular ligule itself instead of the dorsal cirrophore in Stenoninereis species (see [90]).The division between the dorsal ligule and the dorsal cirrophore appears as an intermediate constriction that runs typically from the dorsal cirrostyle towards the base of the dorsal ligule (see [4]: Fig 1C).
Epitokal morphology.Epitokous nereidids are divided in two (pre-natatory and natatory) or three (also post-natatory) body regions, whose reproductive morphological features and their function have been largely addressed in much detail in the literature [1, 5,6,[110][111][112].Interestingly, the epitokes of Pectinereis gen.nov.have body divided into four regions: prenatatory, natatory, post-natatory, and pre-pygidial.During epitoky, chaetigers of the posterior end show less modification than medial segments and are the last to start a transformation process, if at all, because they may present slight changes or remain unmodified [112,113].This also occurs with the post-natatory chaetigers of Pectinereis gen.nov.; parapodial ligules become shorter and cirrostyles elongated posteriorly.However, another distinct body region can be seen between post-natatory chaetigers and pygidium, here referred to as pre-pygidial.Chaetigers of this region are evidently narrower than post-natatory ones, with most of the parapodial projections markedly reduced, barely noticeable, and the dorsal cirrostyles were all detached.Also, the ventral ligules are short and the ventral cirrostyles very much more elongated than in previous chaetigers; however, the most strikingly novel feature of this region is the presence of hooked aciculae (see below).Pectinereis gen.nov. is the only nereidid with a fourth body epitokal region, and it makes the genus unique within the family.
Aciculae.These supportive chaetae in nereidids are deeply embedded within the parapodia so that only sometimes its small tip emerges from the body surface.The typical aciculae have a billiard cue shape-gradually tapering towards the distal end, straight, slender, with a truncate proximal end-sometimes slightly curved, particularly in epitokous parapodia; however, in mature Tambalagamia fauveli Pillai, 1961 they are sharply curved at the tip with a marked sickle shape in natatory chaetigers [6].
Remarkably, some of the aciculae in Pectinereis gen.nov.differ from all the forms previously recorded in nereidids.Although the aciculae present are mainly of the typical form, those shown in the notopodia and neuropodia of most posterior chaetigers (pre-pygidial region), located just immediately before the pygidial rosette-a sex-specific epitokal structure for releasing the sperm through developed papillae [112,114,115]-have a more robust and stouter appearance with a falcate distal end, exhibit a curved body that tapers towards a blunt proximal end, and protrude conspicuously beyond the parapodial surface.All these features of the 'hooked aciculae' are generally more pronounced in the notopodia.They seem to resemble the sickle-shaped aciculae of T. fauveli epitokes, although they differ in form and appearance on the body.For instance, the hooked aciculae in Pectinereis gen.nov. in the post-natatory parapodia are stouter and curved with a falcate distal end, whereas the sickle-shaped aciculae of T. fauveli are slender, straight, with a sharply curved tip, and restricted to the natatory parapodia.
Pectinereis gen.nov.hooked aciculae presumably develop during male sexual maturation before spawning.The appearance of specialized chaetae within epitokous nereidids is a frequent phenomenon.It generally involves the emergence of specific natatory compound chaetae to facilitate swimming within the water column for swarming, which depending on the species, can be paddle-like [112,115,116], capillaries [117] or ensiform [99].However, quite different chaetae have rarely been reported within nereidids at maturity.These are hookshaped non-acicular chaetae ornamented with dorsal spines and forming part of the neuropodial bundle of the third chaetiger in some Micronereis species (see [118]: Figs 9, 25 and 30].These specialized chaetae have been suggested as male genital structures with supporting copulatory functions that are probably used to pierce the epidermis of the posterior dorsal surface of the female for an eventual sperm transfer [118][119][120].Analogous chaetae have been found mainly in the meiofaunal syllid Sphaerosyllis hermaphrodita Westheide, 1990, where the aciculae of a single chaetiger are modified as a solid and straight copulatory structure with a curved and blunt distal end, subdistally broadened and flattened, and ornamented with teeth [121,122]. The function of the hooked pre-pygidial aciculae in Pectinereis gen.nov. is uncertain.Their location and form in the males of P. strickrotti gen.nov., sp.nov., as well as the appearance of modified chaetae in epitokous males of a few nereidids and syllids during reproduction, might suggest some hints on the hooked aciculae function.Epitoke males might produce mechanical body wall ruptures on the fully mature female through the hooked aciculae to discharge the sperm immediately after using the pygidial papillae.Although it is likely that females remain atokous dwelling in the sediment (see below, 'Biology'), successful reproduction in nereidids needs close interaction or contact from a partner of the opposite sex to release the genital products given a chemical stimulus [119,[123][124][125][126][127].When this approach occurs between reproductive individuals of P. strickrotti gen.nov., sp.nov. is unknown.The reliable purpose of hooked pre-pygidial aciculae has yet to be discovered.A detailed study of the reproductive behavior of this species is required.
Ventral cirrophores.Nereidids have ventral cirri consisting of two main components: (1) the distal, usually elongated cirrostyle; and (2) the proximal, slightly or barely developed cirrophore (rarely undeveloped as in Micronereis species, see [118]).These ventral poorly-developed cirrophores are barely noticeable in atokous nereidids or non-natatory regions of epitokes.However, in natatory parapodia of epitokous nereidids, they are typically well-developed as an enlarged and highly vascularized membrane divided into upper and lower lamellae.The upper lamella is generally less developed and may present an additional secondary flap, unlike the single and reniform foliose lower lamellae.
In the pre-natatory parapodia of Pectinereis gen.nov., the ventral cirrophores show the typical non-modified form.Interestingly, in the natatory chaetigers, they are notably different from other epitokous nereidids.The ventral cirrophores are markedly elongated and acuminate, with only a drop-shaped and flattened lower lamella, whose overall form is reminiscent of an elf's shoe.This has not previously been documented within Nereididae.The elfin-shoeshaped cirrophore is assumed here to be an epitokal modification probably used as an oar to move forward during the swimming behavior of males.

Taxonomy
Family NEREIDIDAE de

Etymology
This genus is named by combining the Latin word pectinis (= 'comb') with the name of the type genus of the family, Nereis.The name emphasizes the pectinate (i.e., comb-like) parapodial cirrostyles (gills) in the first anterior chaetigers formed by digitiform filaments.The gender is feminine, as the stem genus-group name.

Remarks
Pectinereis gen.nov.sits well within the subfamily Nereidinae as earlier delineated by Fitzhugh [128] and currently conceived by Alves and colleagues [129].Gills in nereidids are vascularized parapodia structures that have been recorded only in two shallow-water and estuarine genera: Dendronereis Peters, 1854 and Dendronereides Southern, 1921.The gills are modified dorsal cirrophores with multiple filaments starting at least some chaetigers after the first one.However, in the deep-water Pectinereis gen.nov., the gills are modified dorsal and ventral cirrostyles present from the first chaetiger to a few anterior ones.In addition, Pectinereis gen.nov.can readily be distinguished from Dendronereis and Dendronereides by having an anteriorly complete prostomium, two neuropodial (postchaetal and inferior) lobes, and paragnaths only on pharyngeal rings, whereas those two genera have an anteriorly indented prostomium, at least three neuropodial lobes, and papillae only on pharyngeal rings, when present.
In nereidids, the ventral cirrophore is poorly developed compared to the dorsal cirrophore, and this is possibly the reason for having overlooked it in the family's systematics.In some epitokes, however, it is enlarged with additional lamellae but still not distinguished in literature from the cirrostyle, referring to it usually as 'ventral cirrus'.Pectinereis gen.nov. is unique in that it shows an elfin-shoe shaped ventral cirrus in natatory chaetigers, viz., a markedly elongated ventral cirrophore with a lower and drop-shaped lamellae, becoming distally acuminated, where the ventral cirrostyle is attached.Epitoke individuals from other nereidid genera show a short and cylindrical ventral cirrophore in the natatory chaetigers with a reniform lower lamella and, at least, one digitiform upper lamella.
The markedly enlarged dorsal cirrophore is present in the medial and posterior chaetigers of several nereidid genera, whether or not they are in an epitoke stage.For instance, Alitta Kinberg, 1865, Cheilonereis Benham, 1916, Neanthes Kinberg, 1865, Nectoneanthes Imajima, 1972, Nereis Linnaeus, 1758, Paraleonnates Khlebovich & Wu, 1962, Perinereis Kinberg, 1865, and Pseudonereis Kinberg, 1865.Nonetheless, Pectinereis gen.nov.can be distinguished from all those genera because the base of the elongate and fusiform dorsal ligule is attached sub-medially to the enlarged dorsal cirrophore, giving the false appearance of a bifurcate cirrophore.In contrast, the 'A.succinea' species complex and some Neanthes, Nereis, Perinereis, and Pseudonereis species, the dorsal ligule is smaller and shifted toward the distal end of the enlarged dorsal cirrophore, although sometimes it is completely reduced in the posterior chaetigers.On the other hand, in the 'A.virens' species complex, Cheilonereis, Nectoneanthes, and some Paraleonnates species the dorsal ligule is broadly enlarged and embraces entirely the enlarged dorsal cirrophore.Typhlonereis Hansen, 1879 was until now the single nereidid genus exclusive from the deep sea.Over 140 years later, Pectinereis gen.nov. is established as endemic to deep environments.
Proboscis everted, with maxillary and oral rings cylindrical, wider than long (Fig 5A -5C).Proboscis structures observed in holotype only.Jaws slightly crenulate (Fig 5A and 5B), reddish in distal third, remaining amber; with faint traces of 5 short denticles; 2 canals emerging from pulp cavity.
Paragnaths present on both maxillary and oral rings of proboscis, all conical, dark red and brown in maxillary ring, brownish in oral ring (Fig 5A -5C); plate-like basements absent.Area I: 18, five slightly regular rows of uneven cones in broad, triangular patch, medial row cones largest (Fig 5A and 5C).Areas IIa: 26, IIb: 29, two to three regular longitudinal rows of uneven cones in L-shaped patch, distal cones larger (Fig 5A and 5C).Area III: 42, five irregular rows of uneven cones in broad, rectangular patch; proximal and outer cones shorter; 9 and 11 laterally-isolated cones in three slightly regular longitudinal rows (Fig 5B and 5C).Areas IVa: 33 and IVb: 32, three slightly curved longitudinal rows of uneven cones in L-shaped patch, distal cones larger forming one regular, transverse row (Fig 5B and 5C).Pre-natatory region (Fig 6A -6E) with notopodia consisting of dorsal cirri with cirrostyle and cirrophore, dorsal ligule, notopodial prechaetal lobe, and median ligule in biramous parapodia; and neuropodia consisting of neuroacicular ligule with inferior and postchaetal lobes, ventral ligule, and ventral cirrus with cirrostyle and cirrophore (neuropodial superior lobe not developed).

Type locality
Close to summit of Mound 12, near methane seeps at ~1,000 m depth (8.929˚N; 84.313˚W), off the Pacific coast of Costa Rica.

Biology
The swimming worms collected in this study were epitokous males.One of them spawned out all the sperm after removing tissue of most-posterior end for molecular purposes.One incomplete atokous female with well-developed ova (~350 μm diameter) was found dwelling in the sediment (Fig 1C).We can infer that only the males become epitokes and the females remain in the sediment, as occurs in species of Alitta such as A. grandis [6,99,130] and possibly Websterinereis glauca (Claparède 1870) [112].

Distribution
Species known only from type locality.

Discussion
In the present study, we establish a new nereidid genus and species, Pectinereis strickrotti gen.nov., sp.nov., from the deep sea off the Pacific coast of Costa Rica using both atokous and epitokous morphological features and molecular evidence.Additionally, we determined it as a member of the subfamily Nereidinae while assessing its evolutionary position within the family using both mitogenome-scale and multi-gene phylogenetic analysis.The latter result shows no obvious close relatives based on the currently available data.

Embracing epitokal morphology
Several nereidid genera were proposed in earlier literature based on the epitokal morphology [131][132][133][134][135][136], but they were later synonymized as represented reproductive forms of members of other genera [137,138].The use of epitokal morphology within nereidid systematics was largely ignored for a long period.Nonetheless, it has been recently demonstrated that the use of morphological and developmental information from epitokes not only provides valuable information to distinguish at the species level but even enables distinction at broader levels [4,101,136,[139][140][141][142].
For instance, Kainonereis Chamberlin, 1919 and Sinonereis Wu and Sun, 1979 were re-evaluated and distinguished from currently valid genera based on the evident epitokal modifications; for instance, the presence of the elytriform and the napiform dorsal cirri, respectively [140,141].Furthermore, the unique combination of non-reproductive and epitokal patterns of a species previously recognized in Composetia aided in distinguishing the new genus Parasetia Villalobos-Guerrero, Conde-Vela & Sato, 2022 from morphologically similar genera [4].Additionally, Neanthes was strengthened as non-monophyletic considering the broad atoke and epitokal unevenness among its species [101].The relationships within Neanthes had already pointed as polyphyletic in earlier morphological analysis [143], and later confirmed using molecular phylogenetics [15,57].
The presence of two atokal (dorsal cirrostyles as pectinate gills and elongate dorsal ligule emerging sub-medially to enlarged cirrophores) and two epitokal (hooked aciculae and elfinshoe shaped ventral cirrophores) are distinctive features of the remarkable deep-sea nereidid Pectinereis strickrotti gen.nov., sp.nov.Together with the distinct phylogenetic placement, the establishment of a new genus and the description of a new species appears well justified.

Phylogeny and systematics
The phylogenetic reconstructions shown here were inferred from a three-gene analysis (Fig 3) and the complete newly obtained mitogenomes of three species of nereidids plus other sequences retrieved from GenBank (Fig 2B).The latter analysis showed a general agreement with the previous mitogenomic assessment of Nereididae [67], supporting the current nonmonophyletic status of Nereidinae de Blainville, 1818.However, this subfamily is presently the most difficult to diagnose since its membership is broader and much more heterogeneous and speciose than the others.Despite this complexity, Pectinereis strickrotti gen.nov., sp.nov. is regarded within Nereidinae as shown in the topologies of molecular phylogenies and by the overall morphological characters: prostomium with anteriorly entire, biramous parapodia, single ventral cirri, and paragnaths on the pharynx.

Paraleonnates and Laeonereis: Nereidinae or Gymnonereidinae?
The type species of Paraleonnates (P.uschakovi Chlebovisch & Wu, 1962), a supposed member of Nereidinae [see 6], showed a variable position (Figs 2B and 3), as did Laeonereis, which was nested inside Nereidinae (Figs 2B and 3), when it is typically referred to as a member of Gymnonereidinae [see 128].Despite both genera being the subject of recent comprehensive morphological studies [e.g., 106,144], none have dealt with their current subfamily position.
Gymnonereidinae was originally erected by Banse [145] for Gymnonereis Horst, 1919, Ceratocephale Malmgren, 1867, Micronereides Day, 1963 andTambalagamia Pillai, 1961, due to the presence of two unique features among nereidids: (1) the anterior region carrying dense tufts of chaetae, and (2) the double neuropodial cirri.However, Fitzhugh [128] expanded it to include all genera without paragnaths, except Namanereidinae, encompassing thus a more complex group of taxa.Hylleberg & Nateewathana [146] and Khlebovich [6] followed Banse's original concept.Later, Santos et al. [147] restricted Gymnonereidinae sensu Banse [145] to the four original genera through a phylogenetic analysis based upon morphology mainly by the presence of double ventral cirri, dense chaetal bundles in anterior parapodia, and subacicular notopodial chaetae.On the contrary, both Paraleonnates and Laeonereis have single ventral cirri, and the chaetal bundles of the anterior parapodia do not differ significantly in density from the subsequent ones.This different morphology and the molecular tree based on mitochondrial genomes support that none of these two genera belongs to Gymnonereidinae, in which Laeonereis was earlier proposed [see 128] but is currently positioned as sister group to Nereidinae [129, this study].
Paraleonnates consists presently of four species: Paraleonnates bolus (Hutchings & Reid, 1991), P. sootai (Misra, 1999), P. tenuipalpa (Pflugfelder, 1933), and the type species P. uschakovi Chlebovitsch & Wu, 1962.The genus has been typically considered within Nereidinae.Recently, morphology and molecular analysis suggested Paraleonnates does not belong in Nereidinae based on phylogenetic results and in having a Group II mitochondrial gene order, which differs from Group I by the position of three t-RNA genes encoding for tyrosine, methionine, and aspartic acid [61,67,129,147].In the present study, P. uschakovi was placed as the sister group to all other Nereidinae sampled here according to the three-gene tree ( Paraleonnates species exhibit a unique feature among nereidids, namely that the maxillary ring of the proboscis has dorsally transverse rows of paragnaths and papillae.Contrary to the typical features present in the subfamily Nereidinae, Paraleonnates has a set of shared characters that makes its species distinctive: 1) prostomium anteriorly with a deep cleft, 2) glandular oesophageal caeca absent, 3) first two chaetigers with notoacicula, 4) three parapodial lobes present only in neuropodia (superior, inferior, and postchaetal), and 5) falcigers absent throughout.The first three features link Paraleonnates with Ceratonereis Kinberg, 1865 and Solomononereis Gibbs, 1971, two genera related to each other and presently treated as belonging to Nereidinae, although their phylogenetic position within the family is still uncertain-being part of Nereidinae according to morphology [143,147], or doubtfully Ceratonereis as sister to all nereidids based on molecular markers [129].However, Ceratonereis and Solomononereis lack a superior neuropodial lobe, present falcigers, and have different arrangement of paragnaths dorsally on the maxillary ring.On the other hand, the last three features can partially be shared with Alitta, Nectoneanthes, and a few Neanthes species with three neuropodial lobes.Paraleonnates shares with Nectoneanthes both the presence of notoacicula in the first two chaetigers-present in Alitta and absent in those Neanthes species-and the absence of falcigers throughout the body -absent in Alitta and Neanthes species.Nevertheless, Paraleonnates can be easily distinguished from the members of those three genera by the absence of a notopodial postchaetal lobe, the prostomium with an anterior cleft, and the absence of glandular caeca.

Accepting Dendronereidinae
In this study, we also propose to reinstate Dendronereidinae Pillai, 1961 as valid.The subfamily was originally proposed to include the gill-bearing genera Dendronereis Peters, 1854, Dendronereides Southern, 1921, andTambalagamia Pillai, 1961 with the former as type [98].However, as suggested previously [1, 91,145,147] and discussed above, the complex gills in members of those genera are not homologous.They are markedly modified, branched, bipinnate dorsal cirrophores in Dendronereis, arborescent tufts inserted basally between the dorsal cirrostyle and the median ligule in Dendronereides, and foliaceous dorsal cirrophore in Tambalagamia.The latter belongs to Gymnonereidinae due to its close morphological [145,147] and molecular [67,129] similarity to Gymnonereis and Ceratocephale.Also, Dendronereis is distinguishable from Dendronereides by having a ventral ligule (absent in Dendronereides) and in lacking notopodial glandular organs and compound falcigers (both present in Dendronereides).However, molecular studies of Dendronereides have yet to be undertaken.
Members of Dendronereis are unique among nereidids by the presence and type of gills restricted to some anterior chaetigers.Recently, a complete mitogenome was published for Dendronereis chipolini Hsueh, 2019, whose identity was confirmed by a nereidid specialist [66].In the present results (Fig 2 and 3), the placement of D. chipolini was as a sister-group to an apparently misidentified specimen of the species published as Neanthes glandicincta [64], and, based on the sequence similarities, is likely also D. chipolini.The placement of D. chipolini supports the recognition of Dendronereidinae in contrast to its synonymy with Gymnonereidinae as previously proposed [128].
Although Dendronereides was not included in the present mitogenomic analysis, we suggest exclusion from Dendronereidinae because of its marked differences with Dendronereis and leaving it unplaced, resulting in the subfamily being monotypic as earlier proposed by Santos et al. [147].However, its validity still needs to be addressed.Dendronereides is more like Tylorrhynchus-considered as belonging to Gymnonereidinae [128] but recently unplaced (see Figs 2B and 3; [129])-rather than other nereidid genera mainly because of the presence of papillae on both rings of the proboscis, dorsal cirrophores consisting of glandular organs with openings, compound falcigers, and the absence of ventral ligule.These genera require further morphological and molecular data to assess their placement.
Following the recent proposal of revisited definitions for the subfamilies Gymnonereidinae and Nereidinae [129], an emendation of Dendronereidinae is provided below considering the earlier and current knowledge on the morphology of the genus' members [e.g., 1, 91, 148 -151].
Diagnosis (emended from Pillai [98]) Prostomium with anterior cleft.Two antennae.Palps with elongated palpophores and conical palpostyles.Tentacular belt length equal to or longer than length of chaetiger 1. Tentacular cirri with four pairs, distinct cirrophore present.Proboscis without paragnaths, papillae present on both rings; maxillary ring sometimes smooth.Paired esophageal caeca absent.Parapodia biramous, except first two chaetigers, uniramous (lacking notoacicula).Dorsal cirrophore of some anterior chaetigers divided into numerous branchial filaments.Chaetigers with gillbearing parapodia multilobed, including notopodial postchaetal, and superior, inferior, and postchaetal lobes.Dorsal, median, and ventral ligules present.Single dorsal and ventral cirri.Notochaetae and neurochaetae with homogomph spinigers only.Mitochondrial gene order of Group II type as identified by Park et al. [61].[129].New sequences are indicated in bold.Most sequence IDs from BOLD are marked with #. * Laeonereis cf.pandoensis (Monro, 1938) is used here instead of Laeonereis culveri (Webster, 1879) since the specimen was collected in Brazil.One terminal from Alves et al. 2023 [129] was not included here, Ceratonereis longiceratophora Hartmann-Schro ¨der, 1985 as the there was no 16S sequence, the COI sequence (AY583701) is actually a flabelligerid and the 18S sequence appears to be of a hesionid.The correct spelling for sequences lodged on GenBank as Tylorrhynchus heterochaetus is Tylorrhynchus heterochetus.

Composition
Nereidid specimens were repeatedly seen swimming just above the seafloor at ~1,000 m depth off Costa Rica (Eastern Tropical Pacific) on cruises dating back to 2009 (Fig 1 and S1 Video).However, it was not until 2018 that three epitokous males and one fragmented infaunal female were successfully collected near methane seeps of Mound 12 via the submersible DSV (deep submergence vehicle) Alvin, operated from the RV Atlantis (Fig 1B-1D and S1 Video).The

Fig 1 .
Fig 1. Pectinereis strickrotti gen.nov., sp.nov. in life.A, B, D. Several epitokous males swimming near methane seeps of Mound 12 (~1,000 m depth) of the Costa Rica margin and videoed via the submersible DSV Alvin. A. A frame grab from a video taken on Alvin dive 4503 on Feb. 4, 2009.B and D. Frame grabs from video taken on Alvin dive 4987 on Nov. 2, 2018.C. A fragment of an atokous infaunal female was collected at the same depth and locality via sediment pushcore on Alvin dive 4984 on Oct. 30, 2018.A white egg ~350 μm in diameter is visible on the exterior.Scalebar 1 mm.E.An epitokous male swimming near methane seeps of Parrita Scar (~1,000 m depth) of the Costa Rica margin.The specimen was initially caught via slurp with the ROV SuBastian (dive S0218, Jan. 11, 2019) but escaped.Images A, B, D, courtesy of Woods Hole Oceanographic Institute.E, courtesy of Schmidt Ocean Institute.https://doi.org/10.1371/journal.pone.0297961.g001 Fig 2A).The fragment of an atokous infaunal female (Fig 1C) was clearly the same species as the epitokous males (Fig 1A, 1B and 1D) and differed by only 3-5 base pairs.The ML phylogenetic analysis (log likelihood = -39786.879947)based on the concatenated COI, 16S, and 18S DNA dataset of 4,320 bases (Fig 3) showed the new taxon under study here (Pectinereis strickrotti gen.nov., sp.nov.) with no well-supported close relationships among the other Nereididae but was well nested within a strongly supported Nereidinae (Fig 3).It did form a clade with a Hediste terminals but with low support.Paraleonnates was the sister group to Nereidinae, though with moderate support, with Gymnonereidinae as sister group to this clade, though with low support.Tylorrhynchus heterochetus was recovered as sister to all other Nereididae and Namaneridinae and Dendroneridinae forming a grade with respect to the Gymnonereidinae+ Paraleonnates+ Nereidinae clade (Fig 3).
Pectinereis strickrotti gen.nov., sp.nov.specimens show unusual non-reproductive and epitokal morphology among nereidids by the presence of five autapomorphic features.Two of them unrelated to the reproductive modifications: (A) pectinate branchiferous parapodial cirrostyles, and (B) elongate, fusiform dorsal ligule emerging basally to expanded cirrophores.And the other three developed during males epitoky (unknown in females, see species 'Remarks' below): (C) body divided into four regions, (D) hooked aciculae, and (E) elfin-shoe shaped ventral cirrophores.Each of those diagnostic characters makes the new genus unique within the family, as demonstrated in both the morphological (see below) and the phylogenetic analyses (Figs 2B and 3).Hence, a new genus is established and a new species is described.A detailed comparison between Pectinereis gen.nov.and other closely related genera is given in the Remarks section.

Fig 2 .
Fig 2. Haplotype analysis and mitogenome phylogeny.A. Haplotype network of COI data acquired for the three male and one female specimen of Pectinereis strickrotti gen.nov., sp.nov.The holotype sequence (male epitoke) is marked with * and has five base pairs different from the female (infaunal fragment).B. Maximum likelihood (ML) tree derived from analysis of the concatenated 15-gene mitochondrial genome dataset, with the 13 PCGs translated to amino acids.Support values at nodes are bootstrap support percentages after 1,000 pseudoreplicates.https://doi.org/10.1371/journal.pone.0297961.g002

Fig 3 .
Fig 3. Maximum likelihood (ML) tree derived from analysis of nuclear 18S rRNA and mitochondrial COI and 16S rRNA DNA sequences.Support values at nodes are bootstrap support percentages after 1,000 pseudoreplicates.https://doi.org/10.1371/journal.pone.0297961.g003 ); body cream (Fig 4C) with faint traces of brownish pigmentation in palpophore and palpostyles (Fig 5A), and single transverse row of same color on dorsum of preserved specimens, more enhanced on posterior chaetigers.

Fig 7 .Fig 8 .
Fig 7. Distribution of dorsal and ventral branchial filaments of right parapodia from the holotype (SIO-BIC A9836) of Pectinereis strickrotti gen.nov., sp.nov.https://doi.org/10.1371/journal.pone.0297961.g007 Fig 3) or sister to the clade of Tylorrhynchus+ Namanereidinae + Dendronereidinae based on the mitogenomic tree (Fig 2B).At present, we concur with previous studies in not placing Paraleonnates within Nereidinae based on these conflicting results.A critical morphological reassessment of Paraleonnates is needed as well as further molecular data from more taxa.